Methods of regulating uptake and transcellular transport of leukocytes and therapeutics

ABSTRACT

Methods for controlling and regulating engulfment, uptake and/or transcellular transport at a stage following initial engagement of an agent to the endothelium are provided, based on the identification of CAM-mediated endocytosis and the sphingomyelin/ceramide pathway as active steps in transcellular TEM. Administration of regulators relating to the identified pathways, such as NHE1, sphingomyelinases, acid sphingomyelinase and ceramide, permit control and regulation of uptake and transcellular transport. Control and regulation of uptake and/or transcellular transport is applicable in strategies to modulate inflammation, provide controlled and/or targeted delivery of agents, control pathogenic invasion, recover action of an inhibited CAM-mediated uptake or transendothelial pathway, or provide uptake or transendothelial transport by targeting cell surface markers other than ICAM-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/652,165, filed Oct. 15, 2012, which applicationclaims the benefit of U.S. Provisional Patent Application No.61/547,687, filed Oct. 15, 2011. The disclosure of U.S. patentapplication Ser. No. 13/652,165 and U.S. Provisional Patent ApplicationNo. 61/547,687 are hereby incorporated herein by reference in theirentirety, for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 HL098416-01awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of regulating engulfment ordocking structures, vesicular uptake, and transcellular migration ofleukocytes and other agents and relates to methods of utilizingregulated engulfment, uptake, and transcellular endothelial migration.

DESCRIPTION OF THE RELATED ART

Leukocyte recruitment to inflammatory sites requires extravasationacross the vascular endothelium (Ley K, et al. (2007) Nat Rev Immunol7:678-689; Nourshargh S, et al. (2010) Nat Rev Mol Cell Biol11:366-378). This occurs through the sequential steps of white bloodcell (WBC) rolling over endothelial cells (ECs), firm arrest, lateralcrawling, and transendothelial migration (TEM) (Ley et al.; Noursharghet al.). The most studied route of TEM is the paracellular pathway,which involves dissociation of endothelial junctions and leukocyteextravasation between adjacent ECs (Dejana, E (2004) Nat Rev Mol CellBiol 5:261-270; Ley et al., 2007). WBC TEM also occurs via thetranscellular pathway, by crossing the EC body independently of junctionopening (Dvorak, A M et al. (2001) J Histochem Cytochem 49:419-432;Carman C V, et al. (2004) J. of Cell Biol. 167:377-388; Millan J, et al.(2006) Nat Cell Biol 8:113-123), which has been observed in cell culture(Carman et al. 2004; Yang L, et al. (2005) Blood 106:584-592; Millan etal., 2006) and in vivo (Dvorak et al.; Phillipson M, et al. J Exp Med203:2569-2575; Marmon S, et al. (2009) Am J Path 01 1 74:684-692).

The mechanisms regulating these two TEM routes are overlapping and notfully understood (Dejana et al.; Ley et al.; Nourshargh et al.). Theyare both mediated by multiple interactions between molecules on thesurface of the leukocytes and ECs (Ley et al.; Nourshargh et al.). Thisis the case for intercellular adhesion molecule 1 (ICAM-1), atransmembrane glycoprotein of the immunoglobulin superfamily (Muro S(2007) Intercellular Adhesion Molecule-1 and Vascular Cell AdhesionMolecule-1, in Endothelial Biomedicine (Aird W C ed) pp 1058-1070,Cambridge University Press, New York). ICAM-1 is over-expressed on ECsduring inflammation and is an anchor for leukocyte β2 integrinsleukocyte function-associated antigen 1 (LFA-1) and macrophagedifferentiation antigen 1 (Mac-1) (Muro 2007). Through theseinteractions, ICAM-1 is involved in leukocyte firm adhesion to ECs,lateral crawling, and TEM (Phillipson et al.).

ICAM-1 engagement by WBCs mediates signaling involving Ca²⁺, Src kinasesand protein kinase C (PKC), Rho/ROCK-mediated formation of actin stressfibers, and cytoskeleton anchorage to the EC surface via binding ofactin crosslinkers to the cytosolic domain of ICAM-1 (Hubbard A K et al.(2000) Free RadicBiol Med 28:1379-1386; Barreiro O., et al. (2002) J.Cell Biol. 157:1233-1245; Muro 2007). This is key in the formation ofendothelial structures contributing to WBC TEM (Hubbard et al.; Barreiroet al. 2002; Yang L, et al. (2006) Circ Res 98:394-402; Muro 2007; vanBuul J D, et al. (2007) J. Cell Biol. 178:1279-1293). For instance, ECsextend ICAM-1-rich microvilli projections that form a “cup” engulfingWBCs (endothelial docking structure), which depends on the cytosolicdomain of ICAM-1 (Barreriro et al. 2002; Carman et al. 2004; Yang etal., 2005; Oh H-M, et al. (2007) Mol Biol Cell 18:2322-2335; van Buul etal. 2007). Prior to this, leukocytes extend podosomes into shallowinvaginations in ECs in search for sites suitable for transcellular TEM(Carman C V, et al. (2007) Immunity 26:784-797). At these sites,ICAM-1-rich invaginations and vesicles from 200 nm to 1 μm in diametercoalesce, forming transcellular pores of up to 6 μm in diameter, throughwhich leukocytes migrate transcellularly (Carman et al. 2007). Althoughthere is consensus on the key role of ICAM-1 in WBC transcellular TEM,the nature of the transcellular pore and the vesicular pathwayregulating its dynamic formation have not been established. Most workssuggest a contribution of caveolar endocytosis (Millan et al. 2006;Marmon et al. 2009) and/or the related vesiculo-vacuolar organelle(Dvorak et al. 2001). Yet, other works have shown no association orpartial association between ICAM-1 and caveolin-1 in structures thatform during TEM (Carman et al. 2004; Carman et al. 2007).

It is unknown whether both routes of TEM serve similar functions, e.g.,transmigration via the transcellular route may lead to more controlledtransport between the bloodstream and the tissues, whereas theparacellular route involving opening between adjacent ECs may result inmore profound leakage of blood components into tissues and edema. Inthis regard, it is plausible that the transcellular route serves as asurveillance mechanism and/or during physiological (controlled)inflammation, versus the paracellular route which may operate duringinflammatory transmigration and/or pathological (uncontrolled)inflammation.

On the other hand, it is also possible that both routes operate as asurveillance mechanism and/or during the inflammatory response, yet theymay be distinctly used by different types of WBCs (e.g., T or Blymphocytes, neutrophils, monocytes/macrophages, dendritic cells,natural killer cells, etc) and/or in different vascular beds of the body(pulmonary, brain, spleen, liver, skin, joints, the gastrointestinaltract, etc).

In any case, the knowledge of the mechanisms underlying interaction ofWBC with the vascular endothelium as well as the processes regulatingWBC migration across ECs are key for the development of strategies ofprevention, diagnostic, and/or therapy of inflammatory conditions and aplethora of related maladies. In addition, similar mechanisms could beused to facilitate transport of diagnostic agents, therapeutics andtheir carriers to, into, and/or across ECs for translationalapplications.

Thus a need exists in the art for further understanding of thetranscellular TEM pathway and development of methods for controlling andregulating transcellular TEM at a stage following initial engagement ofan agent to the endothelium. Such methods would be useful in strategiesto modulate inflammation as well as to provide controlled delivery oftherapeutics and their carriers in the body.

SUMMARY OF THE INVENTION

The present invention relates to a method of regulating formation ofengulfment or docking structures, uptake or transcellular transport ofan agent, the method comprising administration of a regulator ofCAM-mediated endocytosis or the sphingomyelin/ceramide pathway, whereinsuch administration is effective to regulate engulfment, uptake ortranscellular transport of the agent.

In one aspect, the invention relates to a method of recovery of aninhibited pathway, the method comprising administration of a regulatorof CAM-mediated endocytosis or the sphingomyelin/ceramide pathway,wherein such administration is effective to regulate engulfment, uptakeor transcellular transport of the agent and wherein the inhibition isinhibition of engulfment, uptake or transcellular transport within apathway selected from the group consisting of CAM-mediated endocytosis,sphingomyelin/ceramide pathway, phagocytosis, macropinocytosis,clathrin-mediated endocytosis and caveolar-mediated endocytosis.

In another aspect, the invention relates to a method of potentiatingengulfment, uptake or transcellular migration of an agent, wherein theagent is complexed to a carrier targeted to a non-ICAM cell surfacemolecule or receptor, the method comprising administration of aregulator of CAM-mediated endocytosis or the sphingomyelin/ceramidepathway, wherein such administration is effective to induce uptake ofthe agent and potentiation of transcellular transport of the agent.

In a still further aspect the invention relates to a method ofmodulating inflammation, comprising administration of a regulator ofCAM-mediated endocytosis or the sphingomyelin/ceramide pathway, which iseffective to regulate engulfment, uptake or transcellular transport ofan agent of said inflammation.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the model of Example 1, showing (A) transport ofwhite blood cells (WBCs), lymphocytes (pre-blocked or not) and K562cells across activated HUVEC monolayers and (B) transmigration ofantibody-blocked WBCs in the presence (black bars) or absence (whitebars) of HUVEC monolayer.

FIG. 2 is a graph of the endothelial transmigration (A) and binding (B)of WBCs in the presence or absence of filipin, methyl-β-cyclodextrin(Cdx) or amiloride.

FIG. 3 provides graphs of migrating lymphocytes or K562 cells (lackingICAM-1 and VCAM-1 binding integrins) as described in Example 2, showing(A) transmigration as measured by projecting podosomes into/underHUVECs; (B) location of transmigrated WBCs; and (C) comparison oftransmigrating and arrested WBCs.

FIG. 4 provides graphs of activated white blood cells as described inExample 2, showing (A) transmigration as measured by projectingpodosomes into/under HUVECs; (B) location of transmigrated WBCs; (C)comparison of transmigrating and arrested WBCs; and (D) electronmicrographs showing effect of amiloride on distribution of WBCtransmigration events.

FIG. 5 provides fluorescence micrographs of anti-ICAM beads of Example3, demonstrating (A) engulfment by ICAM-1 rich structure and (B)engulfment by the plasma membrane of the beads and (C) tetraspanin CD9in ECs, at regions of binding of the anti-ICAM beads.

FIG. 6 provides fluorescence micrographs of Example 4, showing molecularrecruitment to sites of endothelial ICAM-1 engagement of anti-ICAMbeads, and modulation of this process.

FIG. 7 provides graphs demonstrating quantification of the moleculesrecruited to the sites of endothelial ICAM-1 engagement of anti-ICAMbeads of Example 4, and modulation of this process.

FIG. 8 provides fluorescence micrographs showing the effects ofamiloride on the formation of ICAM-1-rich endothelial docking-likestructures by anti-ICAM beads, as described in Example 4.

FIG. 9 provides fluorescence micrographs showing distribution ofendothelial ASM upon ICAM-1 engagement by anti-ICAM beads, as describedin Example 5.

FIG. 10 provides fluorescence micrographs showing redistribution ofendothelial ASM and NHE1 upon ICAM-1 engagement, as described in Example5.

FIG. 11 provides fluorescence micrographs showing recruitment ofmolecules at sites of endothelial engagement of a classicalclathrin-associated receptor mannose-6-phosphate receptor (M6PR) byanti-M6PR beads.

FIG. 12 provides electron microscopy images from the in vivo testing ofExample 6, demonstrating presence or absence of CAM-mediated endocytosisin wild type (i-iii) caveolin-1^(−/−) (iv-vi) and ASM^(−/−) (vii-ix)mice.

FIG. 13 provides images of the experiments performed in Example 6; (A)fluorescence micrographs showing formation of ICAM-1-rich vesiclescoalescing into a pore; (B) fluorescence microscopy images showing saidpore surrounded by actin filaments; (C) images showing formation ofpores in the EC underneath a bound particle; and (D) formation oftransmigration pores in binding of WBCs to HUVECs.

FIG. 14 provides the results of the experiments performed in Example 7(A) images showing immunostaining of ASM at the WBC-HUVEC interface; (B)graph of transmigration as measured by projecting podosomes into/underHUVECs; (C) graph of determined location of transmigrated WBCs; and (D)comparison of transmigrating and arrested WBCs.

FIG. 15 provides fluorescence micrographs visualizing the effect ofcoupling acid sphingomyelinase to ICAM-1-targeted particles as describedin Example 8, showing (A) total amount of particles and (B) surfacelocated particles, which enhances uptake of particles by cells deficientin this enzyme.

FIG. 16 provides graphs showing the effects of couplingsphingomyelinases to particles targeted to cell surface markers otherthan ICAM-1, e.g., mannose-6-phosphate receptor (M6PR), where (A)provides percent internalization of said particles prior to coupling toM6PR and in comparison to anti-ICAM particles, and (B) provides dataregarding the uptake of M6PR targeted carriers after coupling to neutralsphingomyelinase to the surface of the carriers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of regulating transcellulartransport of an agent and the involved engulfment or docking structures,and cellular uptake of such agent by manipulation of CAM-mediatedpathways and/or the sphingomyelinase/ceramide pathway and the mechanismsinvolved in such pathways.

ICAM-1 is an immunoglobulin-family transmembrane glycoprotein thatserves as an adhesive surface for leukocytes during inflammation (Yanget al., 2005; Muro et al. 2007; Rothlein R, et al. J Immunol (1986);137:1270-4). It is constitutively expressed on diverse cell types,including, but not restricted to endothelial cells (EC) (Muro et al.,2007; Hopkins, A. M. et al. Adv Drug Deliv Rev 56 (2004): 763-778) andthe expression of ICAM-1 is up-regulated in many pathologies (Muro etal., 2007; Rothlein et al., 1986; Hopkins et al., 2004; Hubbard et al.,2000).

Targeting ICAM-1 to block its adhesive function providesanti-inflammatory benefits (Takei, Y., et al. Transplant Proc 28 (1996):11 03-1 105; Kavanaugh, A. F., et al. Arthritis Rheum 40 (1997):849-853; Hallahan, D. E., et al. PNAS. 94 (1997): 6432-6437). Inaddition, ICAM-1 represents an attractive target for drug delivery todifferent sites in the body. For instance, antibodies to ICAM-1 arebeing explored as therapeutics and affinity carriers in cell cultures,animal models, and early clinical studies, where they have shown goodsafety (Muro et al., 2006; Garnacho, C. et al. JPET 325 (2008): 400-408;Garnacho C, et al. (2008) Blood 11 1:3024-3033; Muro S, et al. (2008)Mol Ther 16:1450-1458; Murciano, J. C. et al. Blood 101 (2003):3977-3984; Muro et al., 2006; Villanueva F S, et al. Circulation 98(1998): 1-5; Weller, G. E., et al. Ann Biomed Eng. 30 (2002): 101 2-1 019 Danilov, S. M. et al. Am J Physiol 280 (2001): L1335-L1347; Sakhalkar,et al. PNAS 100 (2003): 15895-1 5900; Rossin, et al. J. Nucl. Med. 49, 1(2008): 103-111; Muro S, et al. (2005) Blood 105:650-658). The presentinventors have also designed short peptides derived from a naturalprotein present in the human circulation and peptides identified byphage-display, all of which serve as ICAM-1 targeting molecules toprovide efficient and specific binding of therapeutic agents and drugdelivery systems to ICAM-1 in both mice and human cells (InternationalPublication No. WO 2010/141879; U.S. Provisional Patent Applications61/220,404 and 61/184,657).

The involvement of ICAM-1 in leukocyte TEM via both the paracellular andtranscellular routes is known, but its role and the modulation of thispathway are not completely understood. As compared to other endothelialadhesion molecules, ICAM-1 plays a key role in transcellular TEM(Barreiro et al., 2002; Yang et al., 2005; Millan et al., 2006; Ley etal., 2007; Oh P, et al. (2007) Nat Biotechnol 25:327-337). Endothelialendocytic vesicles that form in this process coalesce, generating atranscellular pore through which WBCs transmigrate transcellularly(Carman et al. 2004; Carman et al. 2007). Hence, if ICAM-1 was involvedin the regulation of such transcellular TEM pathway, binding to ICAM-1(ICAM-1 engagement) would contribute to inducing formation of engulfmentor docking structures, endocytic vesicles, and transcellular transportpores in ECs. Specific engagement of endothelial ICAM-1 by other“ligands” (protein conjugates and polymer particles coated withantibodies to ICAM-1 (anti-ICAM) used for drug delivery) is sufficientto elicit formation of endocytic vesicles in cytokine activated ECs(Muro et al., 2003; Muro et al., 2006; Muro et al., 2008).

Cell adhesion molecule (CAM)-mediated endocytosis, distinct fromclathrin- or caveolae-mediated endocytosis, macropinocytosis, orphagocytosis (Muro et al., 2003; Muro et al., 2006; Muro et al., 2008),results in cytoskeletal rearrangement with formation of actin stressfibers (Muro et al., 2003; Muro et al., 2006; Muro et al., 2008). DuringCAM-mediated endocytosis, ICAM-1 interacts with the Na⁺/H⁺ exchangerNHE1, a molecule which acts as a crosslinker of actin filaments to thecytosolic domain of ICAM-1 (Denker, S P, et al. (2000) Mol Cell6:1425-1436; Muro et al., 2006).

Nevertheless, the pathway previously thought to contribute toICAM-1-mediated regulation of leukocyte TEM was the caveolar pathway(Millan et al., 2006; Marmon et al., 2009) and/or the associatedvesiculo-vacuolar organelle (Dvorak et al., 2001). However, formation ofendocytic vesicles upon engagement of ICAM-1 is mediated by CAM-mediatedendocytosis (Muro et al., 2003; Muro et al., 2006; Muro et al., 2008),which is unrelated to said caveolar-mediated endocytosis or the relatedvesiculo-vacuolar organelle (Muro et al., 2003; Muro et al., 2006; Muroet al., 2008). Formation of endocytic vesicles upon ICAM-1 engagement byanti-ICAM particles is not affected by filipin, a drug that sequesterscholesterol in the plasma membrane of cells and inhibitscaveolar-mediated pathways (Muro et al., 2006). Also, anti-ICAM beadsand related vesicles do not co-localize with cholera toxin B, a moleculeknown to bind to ganglioside GM1 in lipid raft-related regions of theplasmalemma, followed by internalization within cells via caveolar- and(alternatively) clathrin-mediated pathways. Indeed, in addition to thecaveolar pathway, none of the other classical endocytic pathways whichnaturally operate in cells in the body, such as said clathrin-mediatedendocytosis, macropinocytosis or phagocytosis, is related to theformation of endocytic vesicles via ICAM-1 engagement (Muro et al.,2003; Muro et al., 2006; Muro et al., 2008).

In addition, transmigration of WBCs across the endothelium is believedto require active regulation by leukocytes (Nourshargh et al 2010).Evidence shows that the ability of leukocytes to migrate laterally alongthe endothelial surface is important in the decision betweenparacellular versus transcellular TEM (Phillipson et al. 2006, Gerard A,et al. (2009) Blood 11 3:6138-61 47), as WBCs probe for sites suitablefor transmigration (Carman et al., 2004). In contrast to the active roleof WBCs interacting with endothelial cells, CAM-mediated endocytosis hasonly been associated with engagement of endothelial ICAM-1 by“artificial” and “inert” objects, such as protein conjugates and polymerparticles targeted to ICAM-1 via anti-ICAM antibodies or peptides (Muroet al., 2003; Muro et al., 2006; Muro et al., 2008), but it has not beenobserved in any of the multiple previous works looking at theinteraction of WBCs with endothelial cells. CAM-mediated endocytosis hasbeen also shown to be induced in the case of engagement of anotherendothelial molecule, platelet-endothelial cell adhesion molecule 1(PECAM-1), by anti-PECAM conjugates and polymer particles (Muro et al.,2006). Yet, only ICAM-1, but not PECAM-1, has been shown to be necessaryfor transcellular TEM of leukocytes.

Also, endocytosis induced by “artificial” and “inert” anti-ICAMconjugates or polymer particles or beads has not been shown to result inendocytic vesicles that coalesce to form transendothelial pores butrather to result in transport of individual vesicles to either plasmamembrane recycling pathways (in rare instances) or (in most instances)to transport to endosomes and lysosomes (Muro et al., 2003, Muro et al.,2005; Muro et al., 2006).

Thus, the role of CAM-mediated endocytosis on WBC TEM, particularly viathe transcellular route, was hardly predictable and somewhat unlikely.Yet, signaling associated to CAM-endocytosis is somewhat similar toleukocytes: Ca²⁺ signaling, activation of Src, PKC, and Rho/ROCK, andformation of actin stress fibers (Muro et al., 2003; Muro et al., 2006;Muro et al., 2008). Also, ECs endocytose anti-ICAM beads of variousshapes and sizes, whose dimensions range from a hundred nanometers toseveral micrometers, both in cell culture and in vivo (Muro et al.,2003; Muro et al., 2005; Muro et al., 2006; Muro et al., 2008) and thisoccurs without opening of the endothelial junctions (Muro et al., 2005;Muro et al., 2008). Therefore, the present inventors explored theassociation among these phenomena in order to more fully understandtranscellular transport.

Using anti-ICAM beads, peripheral blood leukocytes and molecular,cellular and in vivo tools, as detailed in the Examples below, anunexpected role for the sphingomyelin/ceramide pathway commonlyunderlying and connecting these processes was found. The data presentedherein demonstrate that formation of plasmalemma engulfing structures,invaginations and coalescing vesicles, as well as upstream signaling andcytoskeletal restructuring driving CAM-mediated endocytosis of“artificial” ICAM-1 ligands (e.g., anti-ICAM beads) is indeedreminiscent of the events elicited during ICAM-1 engagement by WBCstransmigrating across ECs and contribute to transcellular TEM.

Therefore in one aspect, the invention relates to methods of regulatingtranscellular TEM of agents, involving control of such CAM-mediatedendocytosis and sphingomyelin/ceramide pathway.

Such regulation can be utilized in myriad ways to control cellularuptake, internalization, transport, delivery and/or arrest of agents.Further uses of the regulation include control of pathogens that bind toICAM-1, which may invade within cells or be transported across cells.

In a particular embodiment the invention relates to methods ofregulating transcellular transport of agents, which further provides theability to control the inflammatory interaction of leukocytes and theendothelium and to regulate delivery of therapeutics in the body viaICAM-1 targeting strategies, as well as via binding to other cellsurface markers while providing elements of the CAM- orsphingomyelin/ceramide pathways.

A method of regulating formation of engulfment or docking structures,uptake and/or transcellular transport of an agent is provided, themethod comprising administration of a regulator of CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway, wherein suchadministration is effective to regulate engulfment, uptake ortranscellular transport of the agent.

Initially, the effect of disrupting lipid domains (which have beenassociated to caveolar pathways) or CAM-mediated endocytosis onleukocyte transmigration across endothelial cells was examined A modelfor examination of transmigration of white blood cells acrossendothelial monolayers was developed as described in Example 1. Asdescribed in detail in Example 1 and shown in the results presented inFIG. 2, it was demonstrated that both lipid domains and CAM-mediatedendocytosis are involved in WBC transmigration, but caveolae-mediatedpathways are not.

Example 2 provides a model of paracellular versus transcellulartransmigration of white blood cells, which was used to examine the roleof lipid domains and CAM-mediated endocytosis in paracellular versustranscellular diapedesis of leukocytes. The results of Example 2 suggesta main role for lipid domains and CAM-mediated endocytosis, and notcaveolae-related pathways, in transcellular TEM versus the paracellularroute.

Example 3 documents the evaluation of the association of lipid domainsand the sphingomyelin/ceramide pathway to endothelial docking structuresinduced by ICAM-1 engagement. Endothelial engulfment of WBCs at lipidraft-like, ICAM-1-rich docking structures occurs in association with WBCadhesion to the endothelium and TEM via transcellular pores (Barreiro etal., 2002; Carman and Springer 2004; Oh et al., 2007; van Buul et al.,2007). However, experiments using WBCs involve engagement of multipleadhesion molecules on the endothelial plasmalemma. Example 3demonstrates correlation of ICAM-1 specifically with lipid domains atthe EC surface and formation of engulfing or docking structures.

Example 4 demonstrates that the typical lipid raft-domain componentscholesterol, sphingomyelin and ganglioside GM1 were enriched in areas ofengulfment of anti-ICAM beads in Example 3, mimicking endothelialdocking structures (ring-shaped fluorescent regions in FIG. 6A). Theresults of Example 4 correlate well with the findings presented in FIGS.2 and 4, showing that disruption of lipid domains and CAM-mediatedpathway affect transcellular TEM and indicating that ICAM-1 engagementmay be sufficient to induce endothelial docking structures at theselipid domains.

Since hydrolysis of sphingomyelin into ceramide contributes to formationof large lipid domains and this pathway is associated to modulation ofthe plasmalemma plasticity and cytoskeletal signaling (Holopainen J M,et al. (1998) Biochemistry 37: 17562-1 7570; Zha X, et al. (1998) J.Cell Biol.; 140(1):39-47; Holopainen J M, et al. (2000) Biophys J78:830-838; Zeidan Y H, et al. (2008) J. Cell Biol. 181:335-350),features required during WBC TEM, ceramide was tested for at regions ofICAM-1 engagement by anti-ICAM beads.

As described in Example 4 and shown in FIG. 6C i, iv, ceramide alsoincreased (3.5±0.01-fold) at these regions over adjacent areas, whichwas impaired by EIPA (35.6% decrease; FIG. 6Cii, iv), an amiloridederivative that more specifically inhibits NHE1 involved in CAM-mediatedendocytosis (Muro et al., 2003; Muro S, et al. (2006) Am J Physiol LungCell MolPhysiol 290:L809-817). As such, NHE1 was identified as having acritical function in particle internalization and as a possibleconnector of the CAM-mediated pathway to sphingomyelin/ceramidesignaling.

To investigate this connection further, the potential recruitment ofacid sphingomyelinase during formation of endothelial docking structuresinduced by ICAM-1 engagement was examined, as described in Example 5.Contribution of the sphingomyelin/ceramide pathway to formation ofendothelial docking structures and WBC TEM may require sphingomyelinaseactivity. Given that NHE1 provides acidification at the extracellularside of the plasmalemma (Bourguignon L Y W, et al. (2004) J Biol Chem279:26991-27007), the present inventors theorized that acidsphingomyelinase (ASM), a lysosomal enzyme that can be secreted (JenkinsR W, (2009) Cell Signal 21:836-846), is involved in ceramide generationat these regions.

The results of Example 5 support the assertion that ASM secretion isassociated with the CAM-mediated pathway. Accordingly, Example 6 wasperformed to confirm this by evaluating the effect of impairing acidsphingomyelinase on CAM-mediated endocytosis. Ceramide production by ASMat sites of ICAM-1 engagement where NHE1 acidifies the milieu mayprovide plasmalemma plasticity (and likely cytoskeleton signaling;Holopainen et al., 1998; Zha et al., 1998; Holpainen et al., 2000;Zeidan et al., 2008) required by endothelial docking-like structuresinvolved in engulfment of micron-sized objects and WBCs, which thepresent inventors attribute to the CAM-mediated pathway, rather thancaveolae-mediated pathways.

Furthermore, an in vivo mouse model detailed in Example 6 confirmed thatengulfment leading to endocytosis of anti-ICAM beads by ECs wasinhibited in ASM mice.

Example 7 provides an examination of the role of acid sphingomyelinasein leukocyte transcellular transmigration. The inventors hadhypothesized that ASM was involved not only in the formation ofCAM-mediated vesicles but also fusion of said vesicles into largerstructures, similar to formation of ICAM-1-rich invaginations andvesicles that coalesce into transmigration pores. As such, the examplelooked for ASM to appear at areas of WBC migration across ECs and itsinhibition to affect transcellular TEM.

The results of Example 7 support a concerted role forsphingomyelin/ceramide signaling and NHE1-dependent CAM-mediatedendocytosis induced upon ICAM-1 engagement at the EC surface, intranscellular TEM. This model is consistent with findings obtained usingWBCs and specific ICAM-1 engagement by anti-ICAM beads, pharmacologicalinhibitors and genetically modified models, cell cultures and in vivosystems.

Taken together, the results reported herein support a model fortranscellular TEM that includes, but is not limited to, engagement ofICAM-1 in lipid domains enriched in sphingomyelin, which inducessecretion of acid sphingomyelinase (ASM) from intracellular compartmentsto these areas of the endothelial plasma membrane. At these sites,engaged ICAM-1 forms a complex with NHE1, which results in localacidification, sphingomyelin hydrolysis by secreted ASM, and localproduction of ceramide. This signal leads to actin polymerization andcytoskeleton remodeling, stabilizes the engagement platform byrestricting molecular diffusion and providing cytoskeletal anchorage,regulates membrane deformability, and favors dynamic formation ofCAM-mediated endocytic vesicles, which occurs at sites ofleukocyte-podosome sampling in search from sites suitable fortranscellular TEM. Finally, vesicular fusion mediated throughsphingomyelin/ceramide signaling at this interface results intransmigration pores.

The model of transcellular TEM presented herein explains particularmolecular and cellular features required for such events to take place.For instance, the ion exchange activity of NHE1 regulates the elasticityof the endothelial apical surface (Hillebrand U, et al. (2006)Cardiovasc Res 69:916-924), in agreement with high permissibility ofCAM-mediated endocytosis for engulfment and uptake of large micron-sizedobjects in vitro and in vivo (Muro et al., 2008). This is in contrast tothe caveolar and clathrin pathways, shown to be rather restrictedregarding the size of “ligands” that can accommodate to typical caveolarvesicles (Oh et al., 2007).

Such deformability properties of CAM endocytosis, which can be exploitedfor transport of drug carriers into and across cells, would suitablyadapt for formation of large endothelial docking structures, the widerange of sizes exhibited by the invasive podosomes that leukocytesextend into ECs during TEM, and formation of the transcellular pore(Carman et al., 2004; Carman et al., 2007). Also in the context ofCAM-mediated endocytosis and WBC TEM, diffusion of molecules in theplasmalemma must be temporarily reduced in areas of binding to ICAM-1,to permit formation of engagement and signaling platforms, and to anchorthe cytoskeleton.

The level of deformability required to engulf large objects and cells byendothelial docking structures progressing into transmigration poresmust also relate to a particular lipid composition of the plasmalemma.As shown here, such domains seem to be related also to CAM-mediatedpathway, and are associated to induction of sphingomyelin/ceramidesignaling upon ICAM-1 engagement at the EC plasmalemma. As observed inother systems, ceramide confers particular properties to the membraneenvironment depending on the ratio of raft components (Rotolo J A, etal. (2009) Blood 11 4:3693-3706; Silva L C, et al. (2009) Biophys J96:3210-3222), e.g., it can promote the formation of large lipid domains(Holopainen et al., 1998; Holopainen et al., 2000) or displace lipiddomain constituents to affect membrane function (Zeidan et al., 2008).

Ceramide production by ASM at the outer leaflet of the plasma membranemodifies its curvature and results in vesiculization (Zha et al., 1998;Holopainen et al., 2000; Tam C, et al. (2010) J. Cell Biol.189:1027-1038), as well as cytoskeletal rearrangements (Zeidan et al.,2008). These events downstream of the sphingomyelin/ceramide pathwaycould contribute to formation of large micron-sized vesicles in theabsence of clathrin or caveolin coats, as observed in CAM-mediatedendocytosis (Muro et al., 2003; Muro et al., 2008) (Table 1). Inaddition, ceramide production by ASM is associated with vesicular fusion(Utermohlen 0. et al Immunobiology 21 3:307-314), which could contributeto dynamic formation of transcellular pores from vesicles forming viaCAM-mediated pathway. This agrees with the observation that in ASM^(−/−)mice, the beads that were internalized by ECs were found individually invesicles, while in control mice, vesicular fusion resulted in theaccumulation of several beads within larger compartments (Table 1).

Given that ICAM-1 interacts with NHE1 upon ICAM-1 engagement (Muro etal., 2006), and due to the directionality of NHE1 ion exchange (Na′influx/H′ efflux; Denker et al., 2000; Bourguignon et al., 2004), it isexpected that ion transport activity of NHE1 will locally acidify ICAM-1engagement regions, creating a confined acidic microenvironment. Asimilar function of NHE1 has been shown in the context of otherpH-sensitive enzymes (Bourguignon et al., 2004). Secreted ASM, whose pK,is acidic (Jenkins et al., 2009), would only be able to efficientlyhydrolyze sphingomyelin into ceramide at NHE1-enriched, ICAM-1engagement regions. This provides a suitable explanation for how anacidic enzyme can elicit activity at the otherwise neutral extracellularenvironment and may also contribute to understanding the mechanism bywhich ECs regulate ceramide production with spatial precision. It isalso possible that other sphingomyelinases (e.g., neutral enzymes) mayoverlap with this function.

The discovery of both of the sphingomyelin/ceramide pathway andCAM-mediated endocytosis as key contributors to leukocytetransmigration, particularly via, but not restricted to, thetranscellular route, provides necessary further understanding of thetranscellular TEM pathway and provided an important step in thedevelopment of methods for controlling and regulating transcellular TEMat a stage following initial engagement of an agent to the endothelium.As a result of the discoveries reported herein with regard to themechanisms of leukocyte transmigration, the present invention relates tomethods of utilizing such mechanisms to regulate transendothelialmigration.

Further, varied genetic, biotechnological, or pharmacological (amongother) interventions to regulate (potentiate or inhibit) signalinginduced by ICAM-1, the action of NHE1, ASM or other sphingomyelinases,ceramide production by other methods are provided to promotetransmigration of leukocytes (e.g., to combat infection) or to inhibitsuch transmigration (e.g., to control inflammation). Such methods areuseful to impact a plethora of diseases in which inflammation plays arole, including but not restricted to inflammatory and autoimmuneconditions (rheumatoid arthritis, psoriasis, ulcerative colitis, Crohndisease, etc.), infections and septic shock, ischemia-reperfusioninjury, atherosclerosis and thrombosis, metabolic and genetic disorders,asthma and acute lung injury, cancer and tumor metastasis, and manyothers.

Still further, given that promotion of ceramide production in theplasmalemma (demonstrated herein via a showing of ICAM-1 engagement andthe CAM-mediated pathway activating ASM activity) was shown to provideoptimal conditions for internalization within cells and/or transportacross cells, the discovery of the sphingomyelin/ceramide pathway andCAM-mediated endocytosis as key contributors to transmigration arebroadly applied to support new strategies to control and facilitate bodytransport of diagnostic and therapeutic agents, and their targetingmolecules and carriers. Methods of the invention include intracellulardelivery or delivery across cellular barriers, such as the blood-brainbarrier in the central nervous system, the blood-air barrier in thelungs, the epithelial barrier in the gastrointestinal tract,penetrability into tissues and organ, and the like.

Therefore, in one embodiment the invention relates to a method ofregulating transcellular transendothelial migration of an agent, themethod comprising administration of a regulator of CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway, wherein suchadministration is effective to regulate transcellular TEM of the agent.

As used herein, a “regulator” can be administered to regulate theCAM-mediated endocytosis or the sphingomyelin/ceramide pathway. By“regulation” or “regulating” of CAM-mediated endocytosis or thesphingomyelin/ceramide pathway includes any of controlling, managing,adjusting, directing, manipulating or modulating CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway. Regulators ofCAM-mediated endocytosis or the sphingomyelin/ceramide pathway accordingto methods of the invention are effective after ICAM-1 engagement of theagent. Regulators of the invention target steps of engulfment orformation of docking structures, uptake by cells and formation ofvesicles, as well as pore opening and transcellular TEM or transcytosis.

Administration of a regulator may include actual administration of theregulator in vitro or in vivo to a system or patient in need of suchadministration. Administration may be by any suitable administrationmechanism that provides effective levels of the regulator to theendothelium. Any suitable administrative routes that are compatible withthe selected regulator may be employed. Administration methods ofregulators described herein include, but are not limited to, parenteraladministration, intraperitoneal (i.p.) administration, intravenous(i.v.) administration, intraarterial (i. a.) administration, intradermal(i.d.) administration, intramuscular (i.m.) administration, andsubcutaneous (sc) administration.

Administration of a regulator may also include indirect administrationsuch as induction or inhibition of a regulator within the subject systemor patient.

As exemplified herein, regulators may include, but are not limited to,NHE1, sphingomyelinases, acid sphingomyelinase and ceramide. Furtherregulators useful in methods of the invention may include, but are notlimited to, proteins affecting the CAM-mediated pathway, ICAM-1, lipidsaffecting the CAM-mediated pathway, sphingomyelin, and ceramidases.

Acid sphingomyelinase (ASM), other sphingomyelinases, and NHE1 areproteins that may be directly administered to regulate CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway. Such proteins may beobtained by any means known, from any known source, such as fromorganisms, humans or recombinantly produced. ASM, othersphingomyelinases, and NHE1 may also be administered by indirect means,such as by inducement of expression or over-expression of these proteinswithin the subject system or patient. In one embodiment, methods of theinvention may include gene therapy to induce expression of a regulatorof the CAM-mediated endocytosis or the sphingomyelin/ceramide pathway.In another embodiment, methods of the invention may includeadministration of an activator that activates production of theregulator. Insulin is an activator of NHE1, and other activators includemolecules that activate PKC (e.g., PMA, bryostatin) and Rho, amongothers. Activators of sphingomyelinases are saposins, DC-SIGN, OxPAPC,neutral sphingomyelinase (N-SMase) activation associated factor orNSMAF, molecules that activate PKC, and the like.

Regulators such as ASM, other sphingomyelinases, and NHE1 may also beinhibited by known methods, such as use of siRNA to knock-downexpression of these proteins or by using blocking antibodies. Inductionmay also be achieved by administration of inhibiting compounds.Inhibitors of NHE1 may include, but are not limited to amiloride andderivatives like 5′-(N-ethyl-N-isopropyl)amiloride (EIPA), andbenzoylguanidine (Hoechst type inhibitor (HOE))-type compounds.Inhibitors of ASM may include, but are not limited to imipramine, itsderivatives like desipramine, SR33557, D609, and others. Inhibitors ofsphingmyelinases may include, but are not limited to scyphostatin,3,4-Dichloroisocoumarin Chlorpromazine, Hydrochloride Fumonisin B₁ ,Fusarium moniliforme Gentamycin Sulfate Manumycin A, Streptomycesparvulus N-SMase Inhibitor, GW4869 and N^(α)-Tosyl-Phe ChloromethylKetone. Such inhibition is contemplated within administration of aregulator.

Additional regulators may include, but are not limited to lipids such assphingomyelin and ceramide. As discussed above with regard to proteinregulators, lipid regulators may be administered directly, such as byexogenous application to a system or subject in need of a method of theinvention or may be administered indirectly, such as by modulation ofenzymes involved in the mechanisms of synthesis or degradation routes ofthese lipids. In one embodiment, activation or inhibition of ceramidaseswould degrade ceramide or inhibit its degradation, respectively, andthereby impact the presence of ceramide in the pathway.

Regulators of the invention are useful for regulation of CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway. By such regulation,the engulfment, uptake within vesicles, and/or transcellular transportof an agent can be affected.

In one embodiment of the methods of the invention, the regulatorinhibits CAM-mediated endocytosis and inhibits engulfment, uptake withincells, and/or transcellular TEM or transport of the agent. In anotherembodiment of the methods of the invention, the regulator inducesCAM-mediated endocytosis and induces engulfment, uptake within cells,and/or transcellular TEM or transport of the agent. In still anotherembodiment the regulator inhibits the sphingomyelin/ceramide pathway andinhibits engulfment, uptake within cells, and/or transcellular TEM ortransport of the agent. In a further embodiment, the regulator inducesthe sphingomyelin/ceramide pathway and induces engulfment, uptake withincells, and transcellular TEM or transport of the agent. The inventionfurther contemplates combined effects on CAM-mediated endocytosis andthe sphingomyelin/ceramide pathway.

Agents useful in methods of the invention may include, but are notlimited to, autologous or foreign white blood cells, leukocytes,pathogens, drugs, natural and/or artificial molecules and/or objectsincluding, but not limited to, research, analytical or molecular probes,diagnostic agents, therapeutic agents, biologically active agents,research agents, analytical agents, imaging agents, monitoring agents,enzymes, proteins, hormones, lipids, sugars, nucleic acids,lipoproteins, and chemicals.

Agents may be present alone or may be complexed to an additional moiety.As used herein, “complexed” refers to the association between the agentand the moiety, including binding, fusing, linking, coupling, connectingor otherwise associating the agent and the additional moiety. Theresulting complexes may be a single entity, such as a fusion protein ormay result from coupling via absorption mechanisms, by chemicalmodification, through a cross-linker molecule, or via adaptor molecules.Any such complexing is contemplated in methods of the invention.

Additional moieties for complexing to the agent may include, but are notlimited to, targeting moieties, cargo, carriers, delivery vehicles, andcombinations thereof.

Where the agent is complexed to a targeting moiety, such may include,but is not limited to, a polypeptide such as an antibody, antibodyfragment, single chain Fv derivative, humanized antibody, naturalprotein, peptide, or any other natural, recombinant or syntheticaffinity moiety recognizing CAMs. In other embodiments the targetingmoiety targets a cell surface marker other than ICAM-1, including, butnot limited to, receptors associated to other mechanisms of endocytosisand transport across cells, including but not restricted tophagocytosis, macropinocytosis, clathrin-mediated transport andcaveolar-mediated transport. In one such embodiment the non-ICAMreceptor is M6PR.

Where the agent is complexed to cargo, the cargo may include, but is notlimited to a cell or modified cell, reporter probe, biosensor, marker,antibody, peptide or protein, enzyme, ligand, genetic material (DNA- andRNA-based), drug or chemical, imaging or therapeutic agent, or anycombination of the above. Cargo included in methods of the invention maybe directly delivered by the targeting moiety or may be additionallyassisted by a delivery vehicle or carrier.

The invention provides a new strategy to regulate transcellular TEM andto thereby regulate interaction of leukocytes with the endothelium andtransport thereof and also to regulate transport of therapeutics andtheir carriers in the body, and, still further, to generally regulatecellular uptake of agents within cells and across cells via thetranscellular pathway, supporting multiple basic, research, andtranslational applications. The regulatory methods of the invention arebroadly applicable to methods such as, but not limited to, modulation ofinflammation, pathogen invasion and drug delivery.

In applicability to modulation of inflammation, the identification ofpathways subject to regulation permits control of transcellular TEM. Thetranscellular TEM pathway can selectively be upregulated to promotetransmigration or downregulated to inhibit or avoid transmigration.Further, the methods of the invention can be used to shift betweentranscellular and paracellular transmigration pathways. In paracellularTEM, the “leaky vasculature” of the open junctions between cells canpermit entrance of undesired substance, such as red blood cells,proteins and the like. Accordingly, in some situations it is desirableto favor transcellular TEM. However, in other situations it is desirableto disfavor transcellular TEM.

In other applications, the methods of the invention are useful in thedelivery of agents. Agents may be targeted to a surface marker (e.g.ICAM-1 or a receptor associated to classical vesicular transport,including but not restricted to M6PR) and engulfment, uptake by cells,or transcellular transport downregulated, such that the agent remainsimmobilized on the surface of the EC. As such the agent is anchored onthe EC. Additionally, agents may be targeted to a surface receptor andengulfment, uptake by cells, or transcellular transport upregulated,such that the transport of the agent is controlled. Such steps ofimmobilization and transport may also be combined through use ofmultiple regulators and/or timing of regulators, such that an agent maybe initially immobilized, then transported via transcellular TEM at anappropriate time to a desired locale. The methods of the invention aretherefore applicable to promote or avoid entrance of an agent intoendothelial cells and/or across the endothelial lining.

Still further, the invention relates to applicability of methods of theinvention to control of pathogenic invasion via ICAM-1. Where an agentis a pathogen, the engulfment, uptake within cells, and/or transcellulartransport pathway can selectively be upregulated to promote uptake andtransmigration or downregulated to inhibit or avoid uptake ortransmigration. Promotion of transmigration can be utilized to promoteor otherwise include transport of the pathogens to lysosomes and/orvacuoles for subsequent degradation and protection of the cells in thebody against infection. Inhibition of transmigration can be used toprevent pathogenic cellular invasion.

Further embodiments of the invention provide applicability of themethods of the invention to drug delivery systems. In one embodiment theagent is a drug and such agent is complexed to an imaging agent.Promotion of uptake within cells and/or transcellular transport can beutilized for targeted delivery of the drug to the cells and/or tissues.

In a still further embodiment the invention relates to recovery of theaction of an inhibited CAM-mediated uptake and transcellular transportpathway. In another embodiment, the invention relates to enhancement ofuptake and/or transcellular transport when using targeting to other cellsurface markers and pathways while providing exogenously elements of theCAM- or sphingomyelin/ceramide pathways. The method relates to a methodof recovery of an inhibited pathway, the method comprisingadministration of a regulator of CAM-mediated endocytosis or thesphingomyelin/ceramide pathway, wherein such administration is effectiveto regulate engulfment, uptake or transcellular transport of the agentand wherein the inhibition is inhibition of engulfment, uptake ortranscellular transport within a pathway selected from the groupconsisting of CAM-mediated endocytosis, sphingomyelin/ceramide pathway,phagocytosis, macropinocytosis, clathrin-mediated endocytosis andcaveolar-mediated endocytosis.

Example 8 demonstrates that even in ASM^(−/−) ECs, endocytosis of verylarge objects (anti-ICAM particles about 5 micrometers in diameter) canbe achieved by coupling ASM, as a regulator, to said objects as agents(which in this case improved endocytosis from 7% to 25%). By providingacid sphingomyelinase exogenously to cells where the ICAM pathway hasbeen inhibited, recovery of the pathway action is observed.

Potentially, similar outcomes could be obtained using targeting to othercell receptors even if they do not activate acidification via NHE1, forinstance, by using neutral sphingomyelinases instead of acidiccounterparts.

In a still further embodiment the invention relates to a method ofpotentiating cellular engulfment, uptake and/or transcellular transportof an agent, where the agent is complexed to a carrier targeted to anon-ICAM cell surface molecule or receptor, the method comprisingadministration of a regulator of CAM-mediated endocytosis or thesphingomyelin/ceramide pathway, wherein such administration is effectiveto induce uptake of the agent and potentiation of transcellulartransport of the complex.

As an example of one embodiment of the invention, FIG. 16 demonstratesenhancement of cell uptake of drug delivery carriers bysphingomyelinases. FIG. 16A illustrates the observed percentinternalization into human vascular endothelial cells (HUVECs) at 30minutes or 3 hours, of model 1 micrometer diameter polymer (polystyrene)drug carriers, targeted to either ICAM-1 or mannose-6-phosphate receptor(M6PR). It is seen that the ICAM-1-mediated pathway results in moreeffective uptake of carriers by cells, since ICAM-1 mediates uptake viacell adhesion molecule (CAM) endocytosis, which associates to thesphingomyelin/ceramide pathway, where acid sphingomyelinase regulatesformation of plasma membrane engulfment structures and remodeling of thecytoskeleton, conducive to uptake of objects, even those that aremicrometers in size. In contrast, M6PR is known to mediate uptake viathe clathrin pathway, which lacks the ability to associate with thesphingomyelin/ceramide pathway. FIG. 16B shows improvement of the uptakeof nanocarriers targeted to M6PR by coupling neutral sphingomyelinase tothe surface of these carriers, which modulates uptake by providing thiseffector exogenously.

By coupling exogenous sphingomyelinase as a regulator to drug carriersas complex, uptake of those carriers by cells is enhanced. In the caseof FIG. 16, enhanced uptake within cells by targeting M6PR (associatedto classical endocytosis, in particular clathrin-mediated transport) isobserved. Therefore, in one embodiment, the method of modulating thetranscellular endothelial transmigration includes enhancement of uptakeof cells generally regulated by non CAM-mediated pathways.

In another embodiment the invention relates to a method of modulatinginflammation, comprising administration of a regulator of CAM-mediatedendocytosis or the sphingomyelin/ceramide pathway, which is effective toregulate engulfment, uptake or transcellular transport of an agent ofsaid inflammation.

The advantages and features of the invention are further illustratedwith reference to the following examples, which are not to be construedas in any way limiting the scope of the invention but rather asillustrative of various embodiments of the invention in specificapplications thereof.

Example 1 Model of Endothelial Migration

A confluent EC monolayer from human umbilical vein ECs was grown on aporous membrane through which peripheral lymphocytes isolated fromhealthy individuals (wild-type WBCs) can transmigrate, driven by thepresence of the chemoattractant SDF1-α in the chamber underneath theECs. Under control conditions, 77.7±2.3% wild-type WBCs underwenttransmigration by 30 min, which was abrogated when K562 cells lackingthe ICAM-1- and VCAM-1-binding integrins LFA-1 and VLA-4, respectively,were used (FIG. 1A). Transport of either peripheral blood lymphocytes(control) or K562 cells lacking ICAM-1- and VCAM-1-binding integrins ofwhite blood cells (WBCs) across activated HUVEC monolayers assessed at37° C. by counting WBCs in the bottom chamber underneath HUVEC, 30 minafter adding WBCs to the upper chamber above HUVECs (black bars).Transmigration was alternatively performed prior (control) or afterblocking integrins LFA-1 or VLA-4 on peripheral blood lymphocytes usingmonoclonal antibodies. The horizontal dashed line in FIG. 1A representsWBC transmigration at 4° C.

TEM was also inhibited by blocking LFA-1 or VLA-4 on WBCs usingantibodies (40.8±11.9% or 58.8±5.6% of control, respectively; FIG. 1A)without affecting WBC capacity to transverse the porous membrane in theabsence of ECs (FIG. 1 B). FIG. 1B shows the results of transmigrationof antibody-blocked WBCs in the presence (black bars) or absence (whitebars) of HUVEC monolayer, as a control. Data are normalized to controlvalues, and represent mean and standard errors of the mean (n≧3experiments). *, P≦0.001 by Student's t test. These data, consistentwith previous reports (Shaw S K, et al. (2001) Am J Path 01159:2281-2291; Allingham M J, et al. (2007) J. Immunol. 179:4053-4064),validate the model.

Further, transport of activated peripheral blood lymphocytes (WBCs)across activated HUVEC monolayers was assessed at 37° C. by countingWBCs in the bottom chamber underneath HUVEC 30 min after adding WBCs tothe upper chamber above HUVECs (FIG. 2A black bars). Absence of HUVECmonolayer was a control (FIG. 2A white bars). Transmigration wasperformed in the absence (control) or presence of filipin,methyl-β-cyclodextrin (Cdx) or amiloride. In accord with previous works(Tilghman R W, et al. (2002) FEBS Lett 525:83-87; Barreiro 0, et al.(2005) Blood 105:2852-2861; Millan et al., 2006), WBC transmigration wasinhibited by methyl-β-cyclodextrin (Cdx; 48.1±4.8% of control; FIG. 2A),an agent that depletes cholesterol from cells. However, filipin, a drugthat binds to cholesterol and affects caveolae-mediated pathways, didnot inhibit WBC transmigration (88.0±7.7% of control). Instead,amiloride, which affects Na⁺/H⁺ exchangers (Kleyman T R, et al. (1988) JMembrBiol 105:1-21) and inhibits CAM-mediated endocytosis (Muro S, etal. (2003) J Cell Sci 11 6:1599-1609), reduced WBC transmigration evento a greater extent than Cdx (38.6±3.7% of control; FIG. 2A).

Binding of activated WBCs (pre-stained with green fluorescent calcein)to activated HUVECs growing on glass coverslips was determined afterco-incubation for 30 min at 37° C. in control media or media containingfilipin, Cdx or amiloride, followed by fluorescence microscopy (FIG. 2Bblack bars). The horizontal dashed line represents binding of negativecontrol K562 WBCs, which lack ICAM-I- and VCAM-1-binding integrins. Dataare normalized to control values, and represent mean and standard errorsof the mean (n≧3 experiments). *, P≦0.001 by Student's t test. NeitherCdx nor amiloride impaired the capacity of WBCs to transmigrate acrossthe porous filter in the absence of an endothelial monolayer (FIG. 2A),or affected WBC binding to ECs (FIG. 2B).

Example 2 Model of Transcellular Vs. Paracellular Transmigration ofWhite Blood Cells

The role of lipid domains and CAM-mediated endocytosis in paracellularversus transcellular diapedesis of leukocytes was examined. Todistinguish between paracellular and transcellular TEM, WBCs werelabeled with green-fluorescent calcein and diapedesis was evaluated byfluorescence and scanning electron microscopy as WBCs projectingpodosomes (versus round-shaped WBCs) into or under ECs growing overglass coverslips (FIG. 3; FIG. 4).

FIG. 3 shows the results of the migration of activated peripheral bloodlymphocytes (control) or K562 cells lacking ICAM-1- and VCAM-1-bindingintegrins (pre-stained with green fluorescent calcein), determined afterco-incubation for 30 min at 37° C. with activated HUVECs and analyzed byfluorescence microscopy after fixation. FIG. 3A shows WBCs projectingpodosomes into/under HUVECs, scored as transmigrating (black bars)versus non-transmigrating WBCs, scored as arrested cells (round-likeWBCs; white bars). FIG. 3B shows spatial distribution of transmigratingWBCs, scored as occurring at either the endothelial cell (EC) border(white bars) or center (black bars), measured at <3 μm or ≧3 μm distancefrom the cell border, respectively. FIG. 3C shows transmigrationactivity of WBCs at the EC center (black bars), scored as in FIG. 3A,compared to non-transmigrating activity (arrested; white bars) at theseareas. Data are mean and standard errors of the mean (n≧30 WBCs).

FIG. 4 shows the results of the migration of activated WBCs (pre-stainedwith green fluorescent calcein) incubated over activated HUVECs growingon glass coverslips, determined after co-incubation for 30 min at 37° C.in control media or media containing filipin, methyl-β-cyclodextrin(Cdx) or amiloride, and analyzed by fluorescence microscopy afterfixation. FIG. 4A shows WBCs projecting podosomes into/under HUVECs,scored as transmigrating (black bars). FIG. 4B shows spatialdistribution of transmigrating WBCs scored as occurring at either theendothelial cell (EC) border (white bars) related to paracellulartransmigration or center (black bars) related to transcellulartransmigration, measured at <3 μm or ≧3 μm distance from the cellborder, respectively. FIG. 4C shows transmigration activity of WBCs atthe EC center (black bars), scored as in FIG. 4A, compared tonon-transmigrating activity (arrested, round-like WBCs; white bars) atthese areas. Data are normalized to control values (horizontal dashedlines) and represent mean and standard errors of the mean (n≧30 WBCs).*, P≦0.05; **, P≦0.01; ***, P≦0.001 by Student's t test.

Scanning electron micrographs showing the effect of amiloride ondistribution of WBC transmigration events (FIG. 4D). White arrowsindicate WBCs transmigrating at the EC border. Arrowheads mark WBCstransmigrating (white arrowheads) or arrested (black arrowheads) at ECcenter regions. Nu=Nucleus. Scale bar=10 μm.

In agreement with previous results and published data (Shaw et al. 2001;Yang et al. 2005; Allingham et al. 2007), 69.3±2.3% control versus4.0±4.0% K562 WBCs transmigrated in this model (FIG. 3A; FIG. 4D). Whilethe few transmigration events observed for K562 WBCs were associatedonly to the paracellular cell-cell border, control WBCs migratedsimilarly via the paracellular versus transcellular route (46.6±4.0% and53.4±4.0%, respectively; FIG. 3B; FIG. 4D). All K562 WBCs located inregions away from the cell-cell border (EC center) were arrested, while71.2±3.5% of control WBCs located in these areas underwent diapedesis(FIG. 3C and FIG. 4D).

As set forth in FIG. 2, Cdx and mainly amiloride, but not filipin,inhibited WBC TEM (80.5±6.8%, 63.8±7.3% and 108.7±8.6% of control; FIG.4A). Cdx and amiloride shifted paracellular TEM over transcellular TEM(124.9±17.1% over 78.3±14.9% for Cdx and 139.6±34.1% over 65.5±29.7% foramiloride; FIG. 4B, D). Regarding WBCs located away from the EC borders,Cdx and amiloride decreased diapedesis (71.5±14.6% and 56.0±27.5% ofcontrol) and increased the amount of arrested WBCs (170.5±36.2% and209.1±68.1% of control; FIGS. 4C-D).

Example 3 Study of Formation of ICAM-1-Mediated Engulfement or DockingStructures

Polymer beads coated with multiple copies of an antibody against ICAM-1(anti-ICAM beads) were used. These have been previously used forstudying aspects of leukocyte transmigration (Allingham et al., 2007;van Buul et al., 2007; van Buul et al., 2010) and CAM-mediated pathway(Muro et al., 2003; Muro et al., 2005; Muro et al., 2006; Muro et al.,2008).

Activated HUVECs were incubated with anti-ICAM beads for 15 min at 37°C. to engage ICAM-1 on endothelial cells (ECs), followed by washing andfixation. FIG. 5A shows fluorescence micrographs obtained at differentfocal planes along the z-axis (i to iv, where iv is closest to theplasma membrane), after staining anti-ICAM on the surface of beads usinga FITC-labeled secondary antibody and ICAM-1 on the EC surface using aTexas Red-labeled antibody. Scale bar=5 μm. The micrographs showed that,within 15 min incubation, anti-ICAM beads (immunostained in green FITC)bound to ECs and were engulfed by ICAM-1-enriched membrane protrusions(immunostained in Texas red).

FIG. 5B provides a scanning electron micrograph of an anti-ICAM beadbeing engulfed (arrows) by an EC. Scale bar=2.5 μm. These micrographsconfirm that bead engulfment areas were morphologically similar toendothelial docking structures observed during WBC TEM (Barreiro et al.,2002; Carman and Springer 2004; Barreiro et al., 2005).

FIG. 5C provides fluorescence immunostaining of tetraspanin CD9 in ECs,at regions of binding of anti-ICAM beads. (i) Micrograph showing CD9enrichment as ring-like structures (arrows). Scale bar=10 μm. (ii) CD9fluorescence intensity plot at the mid cross-section plane of anti-ICAMbeads. Data are mean and standard errors of the mean (n≧100 beads).These micrographs also validate the model, where analysis of thefluorescence intensity at the bead mid cross-section region showed thatsites of bead engulfment by ICAM-1 engagement were enriched intetraspanin CD9 (FIG. 5C), as reported for WBCs (Barreiro et al., 2005).

Example 4 Recruitment of Molecules to Sites of Endothelial ICAM-1Engagement

Recruitment of molecules at sites of endothelial ICAM-1 engagement byanti-ICAM beads was investigated and quantified. Activated HUVECs wereincubated with anti-ICAM beads for 15 min at 37° C. to engage ICAM-1 onendothelial cells (ECs), followed by washing and fixation. FIGS. 6A and7A show the results of cholesterol (i), sphingomyelin (ii) organglioside GM-1 (iii), stained using fluorescent blue filipin, greenBODIPY-sphingomyelin, or Texas-red cholera toxin B, respectively. FIGS.6B and 7B show the effect of methyl-β-cyclodextrin (Cdx) on enrichmentof cholesterol labeled with blue filipin (i) or ICAM-1 immunostainedwith a Texas-red-labeled antibody (ii-iii) in regions of anti-ICAM-beadbinding. FIG. 6C shows immunostaining of ceramide using a Texas-redlabeled antibody in regions of anti-ICAM-bead binding in control (i),EIPA-treated (ii), and imipramine-treated (iii) cells. Left panels showfluorescence micrographs and phase-contrast insets of bound beads. Rightpanels show pseudocolored fluorescence intensity reconstructions ofmolecules in the EC plasmalemma at areas of bound beads, which areindicated by arrows on their respective left panels and insets. Scalebar=20 μm. (iv) Ceramide fluorescence intensity plots at the midcross-section plane of anti-ICAM beads bound to ECs under control(circles), imipramine (triangles), or EIPA (squares) conditions. Dataare mean and standard errors of the mean (n≧150 beads) in FIG. 6. Datarepresent mean and standard errors of the mean (n≧65 beads) in FIG. 7.

Fluorescence intensity of cholesterol, sphingomyelin and ganglioside GM1was increased by 1.6±0.04-fold, 3.1±0.1-fold, and 1.4±0.1-fold,respectively, in regions of bead engulfment by ICAM-1 engagement overadjacent areas (FIG. 7A). Cdx treatment to chelate cholesterol(confirmed in FIG. 6Bi and FIG. 7Bi) decreased ICAM-1 enrichment inareas of bead engulfment (43% decrease; FIG. 6Bii-iii; FIG. 7Bii-iii).

Similarly, treatment with amiloride to inhibit CAM-mediated pathwaydecreased ICAM-1 enrichment in engulfment protrusions (22.9% decrease).Fluorescence micrographs of FIG. 8 show activated HUVECs incubated for15 min at 37° C. with anti-ICAM beads to engage ICAM-1 on endothelialcells (ECs), under control conditions (top panel) or in the presence ofamiloride (bottom panel). Cells were washed and fixed, ICAM-1 wasimmunostained using a Texas-red-labeled antibody, and samples wereanalyzed by fluorescence microscopy (right panels) and phase contrast(left panels). Presence or absence of bead engulfment is marked witharrows or arrowheads, respectively. Scale bar=10 μm.

Additionally, ceramide was tested for at regions of ICAM-1 engagement byanti-ICAM beads.

Example 5 Study of ASM Association with the CAM-Mediated Pathway

As shown in FIG. 6 of Example 4 above, imipramine, a drug that inhibitsacid sphingomyelinase (ASM), impaired ceramide enrichment in areas ofbead engulfment associated to ICAM-1 engagement (23.6% decrease; FIG.6Ciii-iv), implicating for the first time ASM in ICAM-1-driven formationof endothelial docking-like structures.

Immunofluorescence of ASM in ECs showed that, in absence of ICAM-1engagement by anti-ICAM beads, most ASM located to vesicularcompartments (likely lysosomes) in the perinuclear region of cells(41.8±4.7 vesicles/cell), while only a few ASM-positive vesicles werefound outside the perinuclear area (23.7k3.3 vesicles/cell; FIG. 9).Activated HUVECs were incubated in the absence (FIG. 9; top panel) orpresence (FIG. 9; bottom panel) of anti-ICAM beads for 30 minutes at 37°C. Cells were fixed and permeabilized, and ASM was immunostained inTexas-red. Arrowheads mark ASM at the perinuclear region of cells.Arrows mark ASM at the cell periphery. Dashed lines mark the cellborders, as observed by phase contrast. Scale bar=10 μm.

Activated HUVECs were incubated with anti-ICAM, anti-VCAM or anti-M6PRbeads for 15 minutes at 37° C. to engage these molecules on endothelialcells (ECs), followed by washing and fixation. Fluorescenceimmunostaining of ASM (FIG. 10A; bottom panels) in regions of respectivebead binding (FIG. 10A; phase-contrast, top panels). FIG. 10B providesfluorescence microscopy showing immunostaining of ASM (green), andICAM-1 (red, i-iv) or NHE1 (red, v-viii). Boxes indicate the respectivebeads and bead regions selected for enlargement in iii and vii, and iniv and viii. Scale bars=10 μm, 2 μm, or 0.5 μm, as indicated. ICAM-1engagement by anti-ICAM beads lead to appearance of ASM-positivevesicles at the cell periphery (1.7-fold and 2.3-fold increase at 15 minand 30 min), and bead engulfment areas became enriched in ASM (FIG.10A). As negative controls for ICAM-1 specificity, beads coated withantibodies to VCAM-1, also involved in WBC TEM, or mannose-6-phosphatereceptor (MGPR), involved in clathrin-mediated transport of ASM(Willingham M C, et al. (1981) PNAS USA 78:6967-6971), did not elicitASM recruitment (FIG. 10A).

Activated HUVECs were incubated with anti-mannose-6-phosphate receptor(M6PR) beads for 15 min at 37° C. to engage M6PR on endothelial cells(ECs), followed by washing and fixation. Phase contrast (FIG. 11; leftpanels) and fluorescence micrographs (FIG. 11; right panels) wereobtained at the mid cross-section plane of beads after immunostainingICAM-1 (top), NHE1 (middle), or clathrin heavy chain (bottom) in TexasRed. Arrowheads indicate lack of enrichment of the corresponding markeraround beads. Arrows indicate enrichment of the corresponding markeraround beads. Scale bar=10 μm. Anti-M6PR beads induced recruitmentneither of ICAM-1 nor NHE1, but recruited clathrin heavy chain (FIG.11), validating the specificity of this model.

In addition, areas of bead engulfment mediated by ICAM-1 engagement(FIG. 10B) revealed that ASM co-localized well with ICAM-1 and NHE1(85.1±2.9% and 85.3±3.4% co-localization). At high magnification, ASMappeared to distribute within ICAM-1- and NHE1-lined vesicularstructures (FIG. 10 Biv,viii), supporting secretion of ASM associated toCAM-mediated pathway.

Example 6 Role of ASM on CAM-Endocytosis and Related Events

As shown in Table 1, inhibition of ASM with imipramine or NHE1 withamiloride, and Na⁺ depletion to impair Na⁺/H⁺ transport andacidification (but not filipin, which affects caveolae) decreasedendocytosis of anti-ICAM beads by ECs in culture (from ˜7% to ˜60% ofcontrol). This was verified using genetically modified models tocircumvent specificity concerns of pharmacological inhibitors. Using ECsisolated from wild-type versus ASM^(−/−) mice, it was observed that lackof ASM reduced endocytosis of anti-ICAM beads (25% of ECs isolated fromwild-type mice; Table 1).

TABLE 1 Internalization (%) HUVECs Control 100.0 ± 7.6   Amiloride 13.8± 3.3** Imipramine 61.9 ± 6.7** Na⁺ depletion  7.4 ± 2.5** Filipin 107.2± 1.5   MLECs Control 100.0 ± 25.4  ASM^(−/−) 25.4 ± 10.5* Values arenormalized to controls. * and ** indicate p ≦ 0.05 and p ≦ 0.001,respectively. n ≧ 10 micrographs from 2 replicates.

This was further confirmed in vivo by injecting anti-ICAM beads intowild-type (FIG. 12 i-iii), caveolin-1^(−/−) (FIG. 12 iv-vi), orASM^(−/−) (FIG. 12 vii-ix) mice intravenously under anesthesia, andafter 3 h lungs were isolated, perfused, and processed for transmissionelectron microscopy. Arrows in FIG. 12 indicate beads being internalizedin endothelial invaginations. Arrowheads indicate beads fullyinternalized in intracellular vesicles. EC=endothelial cell, VL=(blood)vessel lumen. Scale bars=200 nm or 500 nm, as indicated. The number ofbeads internalized within ECs was quantified from the micrographs. Dataare mean and standard errors of the mean (n≧13 micrographs). *, P≦0.05by Student's t test. As in cell cultures, engulfment leading toendocytosis of anti-ICAM beads by ECs was inhibited in ASM mice but notin caveolin-1 mice (12.2% and 92.8% of wild-type mice).

Additionally, in ASM^(−/−) mice, fewer membrane invaginations weredetected in association with anti-ICAM beads (FIG. 12 i,iv,vii), and thebeads internalized in these mice were individually located withinvesicles (FIG. 12 ix), in contrast to large vesicular structurescontaining multiple beads observed in wild-type and caveolin-1 mice(FIG. 12 iii,vi).

Validating this observation on the role of CAM- andsphingomyelin/ceramide pathways on formation of transmigratory pores, itwas further observed that engagement of ICAM-1 in control ECs usinganti-ICAM particles, carriers and protein conjugates lead to formationof multiple vesicles at the plasma membrane, which coalesced into largepore-like structures supported by or in association with the actincytoskeleton (FIG. 13A-C). This is similar to pores that open across ECsduring transmigration of WBCs (FIG. 13D). Activated HUVECs wereincubated with anti-ICAM conjugates for 15 min at 37° C. to allowengagement of ICAM-1 in the cell surface, followed by washing cells andfixation. ICAM-1 in the surface of the plasma membrane was then stainedusing anti-ICAM and a secondary antibody labeled in Texas red (yellowishin the picture). Cells were then washed and permeabilized to accessinternal compartments. ICAM-1 in internal structures was then stainedwith anti-ICAM followed by a secondary antibody labeled in green FITC.Imaging by fluorescence microscopy permitted to observe multiple smallvesicles enriched in ICAM-1 just underneath the cell surface. Thesevesicles appeared to clealesce or merge in large structures preliminaryto pore formation. A similar experiment with similar result is shown byscanning electron microscopy in FIG. 13B. Activated HUVECs wereincubated with 4.5 μm diameter anti-ICAM particles and imaged by dynamicphase-contrast microscopy. A pore forms in the EC underneath a boundparticle, as shown in FIG. 13C.

Example 7 Study of Acid Sphingomyelinase Association in LeukocyteTranscellular Transmigration

Migration of activated WBCs (pre-stained with green fluorescent calcein)incubated over activated HUVECs growing on glass coverslips, determinedafter co-incubation for 30 min at 37° C. in control media or mediacontaining imipramine, and analyzed by fluorescence microscopy afterfixation.

Fluorescence microscopy revealed enrichment of ASM (immunostained inTexas red) in the interface between ECs and transmigrating peripherallymphocytes (stained with green calcein; FIG. 14A). FIG. 14A shows phasecontrast (left panel) and Texas-red immunostaining of ASM (right andbottom panels) at the WBC-HUVEC interface in control conditions.Nu=nucleus. Scale bars=10 μm or 2 μm, as indicated. This is similar toendothelial docking-like structures formed upon sole engagement ofICAM-1 by anti-ICAM beads, as shown in FIG. 10.

Furthermore, imipramine treatment to inhibit ASM led to a decrease inWBC transmigration (46.6±7.6% of control; FIG. 14B), without affectingWBC binding to ECs (108.0±18.6% of control). In FIG. 14B WBCs projectingpodosomes into/under HUVECs, scored as transmigrating (white bars).

As shown for Cdx and amiloride in FIG. 4, ASM inhibition with imipraminedecreased the level of transcellular TEM, while paracellular TEMincreased (FIG. 14C). In FIG. 14C, spatial distribution oftransmigrating WBCs was scored as occurring at either the endothelialcell (EC) border (white bars) or center (black bars), measured at <3 μmor ≧3 μm distance from the cell border, respectively.

From the WBC population located away from paracellular areas, there wasa decrease in transmigrating WBCs and an increase in arrested WBCs (FIG.14D), suggesting that ASM activity that linked to ICAM-1-engagement andNHE1-dependent CAM-mediated pathway is involved in transcellular TEM ofWBCs. In FIG. 14D, transmigration activity of WBCs at the EC center(black bars) was scored as in (B), compared to non-transmigratingactivity (arrested, round-like WBCs; white bars) at these areas. Dataare normalized to control values (horizontal dashed lines), andrepresent mean and standard errors of the mean (n≧100 WBCs). * P≦0.01 byStudent's t test.

Example 8 Enhanced Transport of Carriers by Cells Via Coupling toSphingomyelinases

As shown in FIG. 15, polystyrene particles (4.5 μm diameter), a modelfor a carrier of diagnostic and/or therapeutic agents, were coated withanti-ICAM to target ECs and ASM, to recover uptake and intracellulartransport of carriers by facilitating endocytosis in ASM ECs, which areotherwise voided of ASM and hence do not support CAM-mediated pathway(as shown in Table 1). Particles only coated with anti-ICAM but not ASMwere used as negative controls for lack of internalization. The cellswere incubated with the particles for 30 min at 37° C. to first allowbinding of particles to ICAM-1 on the cell surface. Then, non-boundparticles were washed off and cells were incubated in control media for1 h at 37° C. to permit potential endocytosis of bound particles. Cellswere finally washed and fixed. Surface-bound non-internalized particleswere immunostained using a secondary antibody labeled with Texas red.This antibody can only bind to anti-ICAM on surface-located beads, whileit can not access particles internalized within the cells.

Samples were analyzed by fluorescence microscopy to visualize totalbeads associated to cells by phase contrast (upper panels) andfluorescence microscopy to visualize non-internalized particles (lowerpanels, arrowheads), from which the percent of internalization ofparticles was calculated. Data represent mean and standard errors of themean (n≧15 ECs). * P≦0.05 by Student's t test. Scale bar=20 μm.

As shown in FIG. 16A, polystyrene particles (1 μm diameter), a model fora carrier of diagnostic and/or therapeutic agents, were coated withanti-ICAM or anti-M6PR, a cell surface marker related to classicalendocytic transport pathway (clathrin-mediated uptake or transcytosis,in particular). Particles targeted to M6PR were not efficient in beingtransported by cells, in contrast to particles targeted to ICAM-1, whentested either at 30 min or 3 h incubation at 37° C. FIG. 16B shows thatincorporation of a sphingomyelinase (neutral sphigomyelinase, inparticular) but not a control protein (IgG) to anti-M6PR particlesenhanced transport by cells by providing this element of the CAM- andsphingomyelin/ceramide pathway. Data represent mean and standard errorsof the mean (n≧15 ECs). * P≦0.05 by Student's t test.

While the invention has been has been described herein in reference tospecific aspects, features and illustrative embodiments of theinvention, it will be appreciated that the utility of the invention isnot thus limited, but rather extends to and encompasses numerous othervariations, modifications and alternative embodiments, as will suggestthemselves to those of ordinary skill in the field of the presentinvention, based on the disclosure herein. Correspondingly, theinvention as hereinafter claimed is intended to be broadly construed andinterpreted, as including all such variations, modifications andalternative embodiments, within its spirit and scope.

What is claimed is:
 1. A method of potentiating engulfment, uptake ortranscellular migration of an agent, wherein the agent is complexed to acarrier targeted to a non-ICAM cell surface molecule or receptor, themethod comprising administering a regulator of CAM-mediated endocytosisor the sphingomyelin/ceramide pathway, said regulator exogenouslymodulating the non-ICAM cell surface molecule or receptor, wherein suchadministration is effective to enhance cellular uptake of the agentand/or transcellular transport of the agent via the non-ICAM cellsurface molecule or receptor.
 2. The method of claim 1, wherein thenon-ICAM cell surface molecule or receptor is associated with amechanism of endocytosis and/or transport across cells other thanCAM-mediated endocytosis.
 3. The method of claim 2, wherein themechanism of endocytosis and/or transport across cells is phagocytosis.4. The method of claim 2, wherein the mechanism of endocytosis and/ortransport across cells is macropinocytosis.
 5. The method of claim 2,wherein the mechanism of endocytosis and/or transport across cells iscaveolar-mediated transport.
 6. The method of claim 2, wherein themechanism of endocytosis and/or transport across cells isclathrin-mediated transport.
 7. The method of claim 6, wherein thecarrier is targeted to M6PR and the regulator is not acidsphingomyelinase.
 8. The method of claim 1, wherein the agent isselected from the group consisting of autologous white blood cells,foreign white blood cells, pathogens, drugs, research probes, analyticalprobes, molecular probes, diagnostic agents, therapeutic agents,biologically active agents, research agents, analytical agents, imagingagents, monitoring agents, enzymes, proteins, peptides, hormones,lipids, sugars, nucleic acids, lipoproteins, and chemicals.
 9. Themethod of claim 1, wherein the agent is a biologically active agent. 10.The method of claim 9, wherein the carrier comprises a polymer.
 11. Themethod of claim 1, wherein the regulator is not insulin.
 12. The methodof claim 11, wherein the regulator is selected from the group consistingof proteins affecting the CAM-mediated pathway, sphingomyelinases,activators of sphingomyelinases, NHE1, lipids affecting the CAM-mediatedpathway, sphingomyelin, ceramide, and ceramidases.
 13. The method ofclaim 12, wherein the regulator is selected from the group consisting ofNHE1, sphingomyelinases and ceramide.
 14. The method of claim 1, whereinthe regulator is administered to a patient in need of suchadministration and the administration of the regulator provideseffective levels of the regulator to the endothelium of the patient. 15.The method of claim 14, wherein the administration is parenteraladministration, intraperitoneal administration, intravenousadministration, intraarterial administration, intradermaladministration, intramuscular administration or subcutaneousadministration.
 16. A method of potentiating engulfment, uptake ortranscellular migration of an agent, wherein the agent is complexed to acarrier targeted to a non-ICAM cell surface molecule or receptor, saidnon-ICAM cell surface molecule or receptor associated with aphagocytosis, macropinocytosis, clathrin-mediated or caveolar-mediatedtransport pathway, the method comprising administering neutralsphingomyelinase in vitro to a system or in vivo to a patient in need ofsuch administration, wherein such administration is effective to enhancethe engulfment, uptake and/or transcellular transport of the agent viathe non-ICAM cell surface molecule or receptor.
 17. The method of claim16, wherein the administration is effective to enhance cellular uptakeof the agent and/or transcellular transport of the agent via thenon-ICAM cell surface molecule or receptor.
 18. The method of claim 16,wherein said neutral sphingomyelinase exogenously modulates the non-ICAMcell surface molecule or receptor.
 19. The method of claim 16, whereinsaid non-ICAM cell surface molecule or receptor is associated with aphagocytosis transport pathway.
 20. The method of claim 16, wherein saidnon-ICAM cell surface molecule or receptor is associated with amacropinocytosis transport pathway.
 21. The method of claim 16, whereinsaid non-ICAM cell surface molecule or receptor is associated with aclathrin-mediated transport pathway.
 22. The method of claim 16, whereinsaid non-ICAM cell surface molecule or receptor is associated with acaveolar-mediated transport pathway.